The proposed research is an extension of NIH grant # RO1-GM42762-11 and will support work to be carried out primarily in the laboratory of Dr. Laszlo Vigh, Institute of Biochemistry, Szeged, Hungary. The parent grant concerns a ubiquitous family of stress proteins, the small heat shock proteins (sHsps), that are proposed to act as molecular chaperones in stressed and diseased cells. The parent grant aims to understand sHsp function in vivo, to identify what cellular components/processes they protect or interact with during stress, and to define further the mechanism of sHsp activity. The requested FIRCA grant will explore an alternative, though not necessarily mutually exclusive hypothesis, that sHsps function to modulate membrane properties, both to ameliorate the rapid changes in membrane fluidity induced by heat stress, and to control Hsp expression. Studies will take advantage of the P.I.'s work establishing both the genetics and biochemistry of Synechocystis Hsp 16.6 and the collaborator's expertise in lipid biochemistry and membrane biophysics. The specific aims of the proposed research are: Aim 1: To determine how sHsps affect the phase state, dynamic parameters, and microdomain ("raft") organization of the thylakoid (and cytoplasmic) membrane in vivo by comparison of these properties in wild type Synechocystis and cells lacking the sHsp or carrying a mutant sHsp. Aim 2: To test if sHsp lipid interactions are correlated with thermoprotection of membrane functions as measured by the rate of whole chain photosynthetic electron transport and of PSII activity (thylakoid) and leakage of ions (cytoplasmic membrane). Aim 3: To define lipid interaction properties of Hsp 16.6, including reversibility of association with lipids, the specificity of interactions with native Synechocystis and synthetic lipids, and the structural features of the protein required for lipid interactions. Aim 4: To test if specific membrane remodelling due to sHsp deletion or mutation alters the temperature of induction and duration of Hsp synthesis, consistent with the "membrane sensing theory" of stress. Results will provide data on which to build a more complete model of the cellular function of sHsps. Funds will provide support for two PhD students in the foreign laboratory, and the interdisciplinary nature of the experiments will greatly expand their theoretical and practical training to ensure their success in future research.